JPWO2017122638A1 - Transformant and method for producing transferrin - Google Patents

Abstract

A method of secreting and producing transferrin with high productivity using a transformant of Schizosaccharomyces pombe, and a transformant suitable for the method. It has Schizosaccharomyces pombe as a host, has a transferrin gene, and a secretory signal peptide gene that is present in the upstream region of the transferrin gene and expresses transferrin bound to a secretory signal peptide that functions in the host. A transformant characterized in that the Gas2 gene has been deleted or inactivated, and a method for producing transferrin, comprising culturing the transformant in a liquid medium and obtaining transferrin from the liquid medium .

Description

  The present invention relates to a method for producing transferrin using a transformant incorporating a transferrin gene using Schizosaccharomyces pombe (hereinafter referred to as “S. pombe”) as a host.

Transferrin (hereinafter also referred to as “TF”) is a type of glycoprotein that binds to Fe ³⁺ ions and is deeply involved in iron metabolism. In particular, human serum transferrin (hereinafter also referred to as “human transferrin” or “hTF”) belongs to the in vivo iron-binding protein family and is involved in in vivo iron transport and metabolism. It is used as an additive to culture media, pharmaceuticals, and DDS transport carriers. TF is a glycoprotein of about 80 kDa having two domains, N-lobe and C-lobe, and is synthesized by translation as an immature protein having a secretory signal peptide at the N-terminus. Thereafter, the secretory signal peptide is secreted outside the cell as a cleaved mature glycoprotein.

  As the most common method of TF production, a method is known in which a full-length recombinant protein is produced using a cultured cell line of mammalian cells such as hamster kidney cells (BHK cells). In addition, S. is a fission yeast. A method using a pombe is known. S. Pombe, unlike Saccharomyces cerevisiae, which is a budding yeast, is close to human cells in cell division and transcription, and does not contain substances that adversely affect the human body. Therefore, S. The method of producing a full-length recombinant protein of TF in pombe is an excellent method for producing TF that is ingested by humans such as pharmaceuticals. For example, in Patent Document 1, S.A. A method is disclosed in which a transformant having a pombe as a host and having the hTF gene introduced is cultured in a liquid medium containing casamino acid, and hTF is efficiently produced and secreted into the liquid medium for recovery.

  On the other hand, in a heterologous protein production system using eukaryotic microorganisms such as yeast, in order to improve the production efficiency of the desired heterologous protein, part or all of the genome part of the host unnecessary or disadvantageous for heterologous protein production is removed. It is known to use improved hosts that have been deleted or inactivated. For example, Patent Document 2 discloses S.I. In Pombe, it is described that the production efficiency of heterologous proteins can be improved by using an improved host in which at least one gene selected from genes encoding a specific protease (protease gene group) is deleted or inactivated. ing.

  Further, the hTF protein can be secreted and produced by producing it with a secretion signal (endoplasmic reticulum transfer signal) peptide added to the N-terminus of the hTF protein. For example, S.M. Like a polypeptide derived from the secretion signal of a precursor of a mating pheromone (P-factor) involved in pombe mating. A structural gene encoding a fusion protein in which a secretory signal peptide recognized by Pombe was fused to the N-terminus of hTF protein was introduced. When the Pombe transformant is cultured, the produced fusion protein is cleaved in the secretion signal peptide in the Golgi apparatus or endoplasmic reticulum, and the hTF protein is secreted into the medium. For example, Patent Document 3 discloses that a secretory signal peptide, a domain, and b domain of PDI1 (Protein disulfide isomerase 1), which is a protein having a molecular chaperone function and localized in the endoplasmic reticulum on the N-terminal side of a target foreign protein. By expressing a partial protein composed of an x domain or a partial protein composed of a PDI1 endoplasmic reticulum signal peptide, an a domain, a b domain, a b ′ domain, and an x domain, . It is described that it is possible to increase the amount of secreted production of foreign proteins in pombe. PDI1 has a c domain including an ER transition signal, a domain, b domain, b ′ domain, x domain, a ′ domain, and ER localization signal (ADEL) in this order from the N-terminus. There is one active center (CGHC) of molecular chaperone activity in each of the a domain and the a 'domain, and all four of the a domain, b domain, b' domain, and a 'domain form a thioredoxin fold.

JP 2011-125281 A International Publication No. 2007/015470 International Publication No. 2013/111754

  The object of the present invention is to It is an object of the present invention to provide a method for secreting and producing TF with high productivity using a transformant of pombe, and a transformant suitable for the method.

The transformant according to the present invention is S. cerevisiae. Pombe is used as a host, and has a TF gene and a secretory signal peptide gene that is present in the upstream region of the TF gene and expresses a TF to which a secretory signal peptide that functions in the host is bound. The Gas2 gene that the host originally has is deleted. Or it is inactivated.
As the transformant, the TF gene is preferably a gene encoding human transferrin.
Further, as the transformant, the TF gene is a mutant TF gene in which a mutation is introduced into a gene encoding a natural TF possessed by a mammal, and the mutant TF gene is at least one site of the natural TF. It is preferably a gene encoding a mutant TF in which an asparagine residue that is an N-linked sugar chain modification site is deleted or substituted with another amino acid residue.
The transformant preferably has at least one protease gene originally possessed by the host deleted or inactivated. The protease gene is more preferably a gene selected from the group consisting of a metalloprotease gene group, a serine protease gene group, a cysteine protease gene group, and an aspartic protease gene group. The protease gene to be deleted or inactivated is at least one gene selected from the group consisting of psp3 gene, isp6 gene, ppp53 gene, ppp16 gene, ppp22 gene, sxa2 gene, ppp80 gene and ppp20 gene Further preferred.
Further, as the transformant, the TF gene is preferably linked directly or indirectly downstream of the gene encoding the secretory carrier protein containing the secretory signal peptide, and the gene encoding the secretory carrier protein is More preferably, it is a fusion gene of a gene encoding the secretory signal peptide portion of the host PDI1, a gene encoding the ab domain portion of human PDI1, and a gene encoding the x domain portion of the host PDI1.

The method for producing TF according to the present invention is characterized in that the transformant according to the present invention is cultured in a liquid medium, and transferrin is obtained from the liquid medium.
As a method for producing the TF, the transformant is preferably cultured in a liquid medium having a pH of 5.5 to 6.5, and the transformant is preferably cultured in a liquid medium containing adenine. It is also preferable to obtain transferrin from the liquid medium after culturing the transformant until the cell density (OD ₆₆₀ ) reaches 100 or more.

  The method for producing a transformant according to the present invention is described in S.A. It is characterized by incorporating a secretory signal peptide gene functioning in the host with a pombe and a TF gene located downstream of the secretory signal peptide gene, and deleting or inactivating the Gas2 gene inherent in the host And

By culturing the transformant according to the present invention, TF can be produced efficiently.
In addition, by the method for producing TF according to the present invention, S.I. Pombe can be used to secrete and produce TF with high productivity.

FIG. 1 shows the structure of the secretory expression vector pSL6PDI1 (SP) Hsabx-hTF (N413QN611Q). FIG. 2 is a CBB-stained image of SDS-PAGE of the culture solution of the A8-hTF (1) strain and the A8-gas2Δ-hTF (1) strain. FIG. 3 shows changes over time in the secretion amount of hTF (N413Q / N611Q) protein in the A8-hTF (1) strain and the A8-gas2Δ-hTF (1) strain. FIG. 4 shows the SDS-PAGE CBB of the purified fraction obtained by purifying the hTF (N413Q / N611Q) protein in the culture supernatant of the A8-hTF (1) strain and the A8-gas2Δ-hTF (1) strain by DEAE chromatography. It is a stained image.

[TF gene, secretory signal peptide gene]
In the present invention, “TF gene” refers to a structural gene encoding a mature TF protein. The mature TF protein is hereinafter referred to as “TF protein”.

  In a cell of a biological species originally having a TF gene, a fusion protein in which a protein containing a secretory signal peptide (secretory carrier protein) and the TF protein are linked is expressed, and the fusion protein is secreted by the endoplasmic reticulum or the Golgi apparatus. The protein is cut off and the TF protein is secreted outside the cell.

  On the other hand, S. is a host in the present invention. Since Pombe does not originally have a TF gene, even if a fusion protein in which a secretory carrier protein and a TF protein are linked is expressed in the host, the secretory carrier protein is not always cleaved in the host cell. Accordingly, the secretory signal peptide and secretory carrier protein in the present invention are preferably secretory signal peptides and secretory carrier proteins other than those originally possessed on the N-terminal side of the TF protein, which function sufficiently in the host. In the present invention, “functioning in the host” (secretory signal peptide) means having a function of secreting a fusion protein expressed in the host as a TF protein outside the host cell.

  In the present invention, the TF protein and the secretory signal peptide or secretory carrier protein may be directly linked, or indirectly linked by a linker consisting of a peptide having 1 to several tens of amino acid residues.

[Transformant]
The transformant according to the present invention is S. cerevisiae. Gas2 gene inherently possessed by the host, comprising a TF gene as a host, a TF gene, and a secretory signal peptide gene that is present in the upstream region of the TF gene and expresses a TF protein bound to a secretory signal peptide that functions in the host. Is deleted or inactivated.

The TF protein encoded by the TF gene of the transformant according to the present invention is a natural TF protein derived from any species (a TF encoded by a TF gene present in the chromosome of a wild-type organism). Protein) or a modified protein of a natural TF protein. Examples of the modified protein include a polypeptide having an amino acid sequence in which one or several amino acids in the amino acid sequence of a natural TF protein are substituted, added, or deleted, and having a TF function. A polymorphism comprising an amino acid sequence having a sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more with the amino acid sequence of a natural TF protein, and having a TF function Peptides are mentioned. The function of TF means a function of binding to Fe ³⁺ ions and binding to a TF receptor on the cell surface to be taken into a cell. Examples of the TF protein encoded by the TF gene of the transformant according to the present invention include natural TF proteins derived from mammals such as humans, monkeys, mice, rats, rabbits, cows, horses, dogs and cats, or modifications thereof. Proteins are preferred, and hTF or modified proteins thereof are particularly preferred.

When the TF protein encoded by the TF gene possessed by the transformant according to the present invention is a modified protein of a natural TF protein, the modification includes, for example, asparagine which is an N-linked sugar chain modification site after translation. Preference is given to point mutations in the amino acid sequence in which at least one or more of the acid residues are deleted or substituted with other amino acid residues. When a protein having an N-linked sugar chain modification site is secreted and expressed, the resulting recombinant protein is modified with a high-mannose sugar chain with a non-uniform length, resulting in reduced uniformity and quality. There is a risk.
When the TF gene possessed by the transformant according to the present invention is a mutant TF gene encoding a mutant TF protein into which a mutation that eliminates the N-linked sugar chain modification site is introduced, the transformant is cultured. Thus, it is possible to secrete and produce a recombinant TF protein having a stable TF function and a uniform quality. For example, the mutant TF gene includes at least one N-linked sugar chain among the two N-linked sugar chain modification sites (432th and 630th aspartic acid residues) of the natural hTF protein. A mutant TF gene encoding a mutant TF protein in which the asparagine residue as a chain modification site has been deleted or replaced with another amino acid is preferred, and the two N-linked sugar chain modification sites are amino acids other than asparagine residues. A mutant TF gene encoding a mutant TF protein substituted with a residue is more preferable, and a mutant TF gene (sequence) encoding a mutant TF protein in which two N-linked sugar chain modification sites are substituted with glutamine residues. Number 2) is more preferred.

  The secretory signal peptide gene possessed by the transformant according to the present invention is a structural gene encoding a secretory signal peptide that functions in the cells of the transformant. The secretory signal peptide gene may be part of a structural gene that encodes a secretory carrier protein. That is, the structural gene encoding the secretory carrier protein may have a structural gene portion encoding a secretory carrier protein other than the secretory signal peptide gene portion downstream of the secretory signal peptide gene. The secretory signal peptide is S. cerevisiae. Anything that functions within the pombe can be used. Those described in International Publication No. 1996/23890 such as a secretory signal peptide of a secretory protein possessed by Pombe and a P3 secretion signal (34 amino acid residues) can be used. Pombe's PDI1 secretion signal peptide is preferred.

S. The secretory carrier protein that functions in the pombe is preferably a full-length protein of PDI1, a partial protein of PDI1, or a protein in which these mutant proteins are fused, including a secretory signal peptide. In particular, a protein containing the a domain of PDI1 or a protein containing the a ′ domain of PDI1 is preferred, and a protein containing at least one of the a domain or a ′ domain of PDI1 and at least one of the b domain or b ′ domain is more preferred. More preferred is a protein comprising at least one of the a domain or a ′ domain, at least one of the b domain or b ′ domain, and the x domain (see Patent Document 3). Each domain of PDI1 contained in the protein fused downstream of the secretory signal peptide may be derived from the same species or a combination of domains derived from two or more species.
As the transformant according to the present invention, the ability to produce TF is further enhanced. A fusion gene in which the secretory signal peptide gene of Pombe PDI1 and a gene encoding the abbx domain part of human PDI1 or the gene encoding the abb'x domain part of human PDI1 are directly or indirectly linked; Pombe PDI1 secretory signal peptide gene and human PDI1 ab domain part gene or human PDI1 abb 'domain part gene A fusion gene in which the gene encoding the x domain part of Pombe's PDI1 is linked directly or indirectly is preferred.

  S. Pombe does not originally have a TF gene. Therefore, the transformant according to the present invention is S. cerevisiae. A TF gene derived from an organism other than Pombe is obtained by genetic engineering. It is obtained by introducing it into Pombe. The base sequence of the TF gene possessed by the transformant according to the present invention may be the gene sequence itself of the biological species from which the TF protein is derived. It may be modified to a codon frequently used in pombe.

  The gene in which the secretory signal peptide gene and TF gene of the transformant are directly or indirectly linked (hereinafter referred to as SP-TF gene) may be 1 copy or 2 copies or more. Good. The SP-TF gene is It may be introduced as a plasmid outside the pombe chromosome. It may be introduced into the pombe chromosome. By introducing a foreign gene into the chromosome, a transformant having excellent passage stability can be obtained.

  Regarding gene recombination methods using yeast as a host, various expression systems, particularly expression vectors, secretion signals, gene transfer expression vectors, and the like have been developed in order to more stably and efficiently express heterologous proteins. These can be widely applied in the production of transformants according to the above. For example, S.M. Examples of expression systems using pombe as a host include Japanese Patent No. 2776085, Japanese Patent Application Laid-Open No. 07-163373, Japanese Patent Application Laid-Open No. 10-215867, Japanese Patent Application Laid-Open No. 10-215867, Japanese Patent Application Laid-Open No. 11-192094, and Japanese Patent Application Laid-Open No. 11-192094. -1919944, JP-A-2000-262284, WO96 / 023890, etc. are known, and these expression systems can be widely used for the production of the transformant according to the present invention.

  S. host The introduction of the SP-TF gene into Pombe is, for example, SP-TF gene and S. cerevisiae. An expression cassette containing a promoter and terminator that functions in a pombe can be introduced into the host cell as it is, or an integrated vector can be introduced into the host cell. The expression cassette may contain any one or more of a 5'-untranslated region and a 3'-untranslated region, and may contain an auxotrophic complementary marker such as ura4 gene.

  S. Examples of promoters that function in pombe include alcohol dehydrogenase gene promoter, nmt1 gene promoter, fructose-1, 6-bisphosphatase gene promoter, invertase gene promoter (see International Publication No. 99/23223), heat shock protein gene Promoters (see International Publication No. 2007/26617, International Publication No. 2014/030644), etc. Examples include promoters derived from animal cell viruses such as Pombe's inherent promoter, hCMV promoter, and SV40 promoter. S. Examples of the terminator that functions in the pombe include an LPI (human lipocortin I) terminator.

  For example, plasmids derived from E. coli such as pBR322, pBR325, pUC118, pUC119, pUC18, and pUC19 are preferably used as the vector into which the expression cassette is incorporated. The vector preferably has a marker for selecting a transformant. Examples of the marker include ura4 gene (auxotrophic complementary marker) and isopropylmalate dehydrogenase gene (leu1 gene).

  An expression cassette containing the SP-TF gene was obtained from S. cerevisiae. When integrating into the pombe chromosome by homologous recombination, the target site into which the expression cassette is incorporated is S. cerevisiae. It may be present only at one location in the pombe chromosome, or may be present at two or more locations. When two or more target sites exist, S.P. The vector can be integrated at two or more positions on the pombe chromosome. When incorporating an expression cassette into one target site, for example, the target site described in the method described in JP-A-2000-262284 can be used. Two or more vectors having different integration sites can be used to integrate the vectors into different target sites.

  As a vector into which an expression cassette containing an SP-TF gene is incorporated, for example, a vector into which a secretory signal peptide gene and an hTF gene described in Patent Literature 1, Patent Literature 3 and the like are incorporated can be used.

The transformant according to the present invention is a host of S. cerevisiae. The Gas2 gene (SPBC29A10.08.1) inherent in Pombe has been deleted or inactivated. As shown in Examples below, S. cerevisiae into which the SP-TF gene has been introduced. When the Pombe transformant is cultured at a high density where the cell density (optical density at 660 nm measured by a turbidimeter: OD ₆₆₀ ) is 100 or more, Gas2 (1,3-β-glucanosyl transferase) is combined with TF. Increased protein expression level. The Gas2 protein is a glycoprotein that is localized in the cell wall and is involved in the biosynthesis of the cell wall of fission yeast, and is not considered to be directly involved in the expression and secretion of TF. Nevertheless, in the high-density cell culture process of the TF expression strain (transformant introduced with the SP-TF gene), the Gas2 protein is secreted in a large amount in the liquid medium simultaneously with the TF protein, and the production of TF Efficiency is reduced. Strangely, the increase in expression and secretion amount of Gas2 protein observed during high-density culture of TF expression strains is not clearly observed during general batch culture with an OD ₆₆₀ of 20-30. Since the Gas2 gene is deleted or inactivated in the transformant according to the present invention, the Gas2 protein is not expressed even during high-density culture, and the amount of TF secreted into the liquid medium is also increased.

  S. The entire base sequence of the Pombe chromosome is recorded and disclosed as “Schizosaccharomyces pombe Gene DB (http://www.genedb.org/genedb/pombe/)” in the database “GeneDB” of the Sanger Institute. As described in S.A. The sequence data of the pombe gene can be obtained by searching from the database by gene name or strain name.

  The transformant according to the present invention is preferably a transformant in which at least one of protease genes (genes encoding proteases) has been deleted or inactivated. S. By inhibiting the protease activity of at least one protease gene inherently possessed by Pombe, the production efficiency of TF protein is improved and the production amount of TF protein is further increased. The transformant according to the present invention includes serine protease gene group (group of genes encoding serine protease), aminopeptidase gene group (group of genes encoding aminopeptidase), carboxypeptidase gene group (encodes carboxypeptidase). Preferably, one or more genes selected from the group consisting of a group of genes) and a dipeptidase gene group (a group of genes encoding dipeptidase) have been deleted or inactivated.

  As a transformant according to the present invention, only one type of protease gene may be deleted, or two or more types of protease genes may be deleted. Among them, metalloprotease gene group (group of genes encoding metalloprotease), serine protease gene group, cysteine protease gene group (group of genes encoding cysteine protease) and aspartic protease gene group (encoding aspartic protease) Preferably, a transformant in which at least one gene selected from the group consisting of genes is deleted is preferable, and at least one gene selected from the group consisting of metalloprotease gene group and serine protease gene group and cysteine A transformant in which at least one gene selected from the group consisting of a protease gene group and an aspartic protease gene group is deleted is also preferable.

S. Examples of the four protease gene groups of Pombe include the following.
Metalloprotease gene group: cdb4 (SPAC23H4.09), mas2 (SPBC18E5.12c), pgp1 (SPCC1259.10), ppp20 (SPAC4F10.02), ppp22 (SPBC14C8.03), ppp51 (SPAC22G7.01c), ppp52 (SPBC18A7 .01), ppp53 (SPAP14E8.04).
Serine protease gene group: isp6 (SPAC4A8.04), ppp16 (SPBC1711.12), psp3 (SPAC1006.01), sxa2 (SPAC1296.03c).
Cysteine protease gene group: ppp80 (SPAC19B12.08), pca1 (SPCC1840.04), cut1 (SPCC5E4.04), gpi8 (SPCC11E10.02c).
Aspartic protease gene group: sxa1 (SPAC26A3.01), yps1 (SPCC1795.09), ppp81 (SPAC25B8.17).

  The transformant according to the present invention is a host strain of S. cerevisiae. It can be produced by deleting or inactivating the Gas2 gene of the transformant in which the expression cassette containing the SP-TF gene has been introduced into the pombe. It can also be produced by introducing an expression cassette containing the SP-TF gene into pombe. As a host used in this case, at least one protease gene has been deleted or inactivated. By using the Pombe mutant, a transformant in which the SP-TF gene has been introduced, the Gas2 gene has been deleted or inactivated, and at least one protease gene has been deleted or inactivated can be obtained. It is done. S. Examples of mutant strains in which at least one protease gene originally possessed by Pombe is deleted or inactivated include psp3 gene, isp6 gene, ppp53 gene, ppp16 gene, such as A8 strain described in Patent Document 2, The ppp22 gene, the sxa2 gene, the ppp80 gene, and the ppp20 gene have been deleted. Pombe mutants are preferably used.

  As a method for deleting or inactivating the Gas2 gene and deleting or inactivating the protease gene, known methods can be used. Specifically, the gene can be deleted by using the Latour method (Nucreic Acids Research, 2006, 34, e11, published in WO 2007/063919). Moreover, the mutation isolation method using a mutation agent (Yeast molecular genetics experiment method, 1996, Academic Publishing Center) and the random mutation method using PCR (PCR Methods Application, Vol. 2, pages 28-33, 1992) etc., the gene can be inactivated by introducing a mutation into a part of the gene. In addition, the part where a specific gene is deleted or inactivated may be an ORF (open reading frame) part or an expression regulatory sequence part. A particularly preferred method is a method of deletion or inactivation by a PCR-mediated homologous recombination method (Yeast, Vol. 14, pages 943-951, 1998) in which the ORF portion of the structural gene is replaced with a marker gene.

  Any known transformation method may be used as the transformation method. Examples of the transformation method include conventionally known methods such as lithium acetate method, electroporation method, spheroplast method, glass bead method, and the method described in JP-A-2005-198612. A commercially available yeast transformation kit may also be used.

The transformant according to the present invention can be cultured in the same manner as natural Schizosaccharomyces yeasts.
As a liquid medium for culturing the transformant, a known yeast culture medium can be used, which contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by a yeast belonging to the genus Schizosaccharomyces. What is necessary is just to be able to culture genus yeast efficiently. As the liquid medium, a natural medium or a synthetic medium may be used.
Examples of the liquid medium include MMA (Minimal medium with agar), SDC (Synthetic dextrose complete medium), TES (0.5% Bacto-yeast extract, 3% glucose, supplemented with uracil, leucine, histidine, lysine, adenine), YES (0.5% Bacto-yeast extract, 3% glucose, supplemented with uracil, leucine, histidine, lysine, adenine), YPD (1% Bacto-yeast extract, 2% Bactopeptone, 2% glucose) and the like.

  Examples of the carbon source include sugars such as glucose, fructose, and sucrose. Examples of the nitrogen source include inorganic acids such as ammonia, ammonium chloride, and ammonium acetate, ammonium salts of inorganic acids, peptone, and casamino acids. Examples of inorganic salts include magnesium phosphate, magnesium sulfate, and sodium chloride. Specifically, nutrient medium such as YPD medium (MDRose et al., “Methods In Yeast Genetics”, Cold Spring Harbor Labolatory Press (1990)) or minimal medium such as MB medium (K. Okazaki et al., Nucleic Acids Res., 18, 6485-6489 (1990)) can be used.

A well-known yeast culture method can be used for culture | cultivation, for example, it can carry out by shaking culture, stirring culture, etc.
Moreover, it is preferable that culture | cultivation temperature is 23-37 degreeC, and it is more preferable that it is 30-32 degreeC. Further, the culture time can be determined as appropriate.
Further, the culture may be batch culture or continuous culture.

[Method of manufacturing TF]
S. When a transformant (TF expression strain) having a SP-TF gene using pombe as a host is cultured in a liquid medium, the TF protein secreted in the liquid medium after the culture can be obtained. The liquid medium and culture method for cultivating the TF expression strain can be carried out by a known culture method using a known yeast culture medium, similarly to the culture of the transformant according to the present invention.

TF protein is easily degraded under acidic conditions. Therefore, in the production of TF, the pH condition of the liquid medium in which the TF expression strain is cultured is preferably pH 5.5 or more, and more preferably pH 5.5 to 6.5. As the culture time becomes longer, the pH of the culture solution tends to decrease. Therefore, in the culture for the production of TF, the pH of the culture solution is appropriately maintained within the range of 5.5 to 6.5. It is preferable to adjust the pH.
Since the amount of secretory production of TF protein can be increased, the culture time for culturing the TF expression strain is preferably 2 days or more, more preferably 3 to 6 days, and further preferably 3 to 5 days.
It is preferable that the liquid medium for culturing the TF expression strain contains adenine. When the TF expression strain has one or more copies of the SP-TF gene, it tends to lyse and the expression level of TF tends to decrease. By culturing in an adenine-containing liquid medium, even a TF expression strain having one or more copies of the SP-TF gene proliferates well, and the secretion efficiency of TF increases. The adenine content of the liquid medium can be, for example, 0.5 to 200 mg / L, preferably 1 to 100 mg / L per OD ₆₆₀ indicating the cell density.

In order to increase the productivity of TF, it is preferable to culture the TF-expressing strain at high density. In particular, the initial medium and fed-batch culture are performed under the conditions of pH 5.5 to 6.5, preferably pH 5.5 to 6.0, while controlling the aeration rate of the culture tank, the stirring rate, and the addition rate of the fed-batch medium. Preferably it is done. Since the medium is continuously added (fed) according to the growth rate and nutrient consumption rate while keeping the glucose concentration of the medium low at a certain level, the bacterial cell density is finally adjusted in a test tube or flask. Compared with batch culture, it is improved by several tens of times, a high cell density can be achieved, and a larger amount of TF can be secreted and produced. In high-density culture of a TF-expressing strain, it is preferable to culture until the OD ₆₆₀ reaches 100 or more, preferably until the OD ₆₆₀ reaches 200-800.

  As the TF expression strain, the transformant according to the present invention is preferable. The transformant according to the present invention has no expression of the host-derived Gas2 protein and has a high efficiency of secretory expression of TF. In addition, when the TF protein is purified from the liquid medium after high-density culture, separation and purification from the Gas2 protein is not required, and purification can be performed more simply.

  A known method is used to obtain the TF protein from the liquid medium after the culture. For example, a method in which bacterial cells are separated and removed from a liquid medium after completion of culture by centrifugation, and the resulting culture supernatant is adsorbed on an affinity column and washed and then eluted, a dye using activated carbon, etc. A method for removing impurities, a method for separation using a separation membrane, and the like are performed. When a TF expression strain having a Gas2 gene is cultured at high density, a large amount of Gas2 protein is contained in the collected culture supernatant, so an anion using DEAE resin is used for the culture supernatant. When exchange chromatography is performed, the fraction containing TF protein also contains Gas2 protein. Therefore, the TF protein can be purified by separation from the Gas2 protein only after the fraction containing the TF protein is further subjected to hydrophobic interaction chromatography or gel filtration chromatography. On the other hand, the TF protein can be purified by performing only one anion exchange chromatography using DEAE resin on the culture supernatant after the high-density culture of the transformant according to the present invention.

  Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples. However, the present invention is not limited by the following description. In this example, “%” means “% by mass” unless otherwise specified.

[Example 1]
<Generation of hTF mutant gene>
The ORF (SEQ ID NO: 1) of the natural hTF gene encodes a protein having a total length of 698 residues in which an hTF protein is fused downstream of a 19-residue secretory signal peptide. Perform PCR using a primer pair (Forward primer # 9220 (SEQ ID NO: 3) and Reverse primer # 9221 (SEQ ID NO: 4)) using the human cDNA library as a template, and 5 'end of the gene encoding the hTF protein. A DNA fragment in which a base sequence encoding 4 residues (Leu-Lys-Lys-Arg) of the C-terminal part of the restriction enzyme site AflII and the abx domain of human PDI1 is added to the 3 'end side and the restriction enzyme site XbaI, respectively. Got.

  A double digestion product of AflII and XbaI of the obtained DNA fragment is arranged with a gene encoding a P3 secretion signal peptide downstream of the hCMV promoter, and a chromosomal single locus of fission yeast having a multiple cloning site between the gene and the LPI terminator By integrating into the double digestion product of AflII and XbaI of pSL6P3 (Patent Document 3), an integrative secretion expression vector, by ligation and transformation into E. coli DH5α, 4 residues of the C-terminal part of the abx domain are left at the N-terminus. Plasmid DNA of an expression vector pSL6P3-hTF in which a gene encoding a natural type hTF protein containing a group was cloned was prepared.

In addition, in the secretory expression vector in which the target protein expression cassette is incorporated into the multicloning site of the pSL6P3 vector, the vector fragment that has been linearized by cutting with the restriction enzyme NotI is adjacent to the fission yeast chromosome by chromosome homologous recombination. Thus, one copy is incorporated between two existing loci leu1-32 and top2. Further, the pSL6P3 vector, the mutant (leu1 ^-) gene complementary point mutations (Leu1-32) portion (leucine synthesis system gene (gene leu1) has been inactivated by point mutation (leu1-32)) A leucine marker gene fragment (leu1 ⁺ ) is incorporated. Because when the leucine auxotrophy yeast containing gene as a host strain, a transformant linearized fragment pSL6P3 vector is integrated into the chromosome to recover from leucine auxotrophy, - Thus, mutant ^(leu1) Clones can be selected on a minimal medium (MMA plate) that does not contain leucine.

  By a PCR method, a mutation that substitutes glutamine residues for the asparagine residues at the 413th and 611st positions (432th and 630th positions of SEQ ID NO: 1) of the natural hTF protein was introduced into the hTF gene in pSL6P3-hTF. PSL6P3-hTF (N413Q / N611Q) was prepared. Specifically, four point mutations were introduced in which the first base of the codon encoding each asparagine residue was replaced with cytosine (C) and the third base was replaced with adenine (A). The sequence of hTF (N413Q / N611Q) into which the mutation has been introduced is shown in SEQ ID NO: 2.

<Preparation of hTF (N413Q / N611Q) expression vector>
A DNA fragment containing the coding region of hTF (N413Q / N611Q) obtained by double digesting pSL6P3-hTF (N413Q / N611Q) with AflII and XbaI was placed in the downstream of the hCMV promoter. Pombe PDI1 secretion signal peptide and human PDI1 ab domain A chromosomal single locus integrative secretion expression vector of fission yeast comprising a gene encoding a secretory carrier protein (PDI1 (SP) -Hsabx) consisting of the x domain of Pombe's PDI1, and a multicloning site between the gene and the LPI terminator pSL6PDI1 (SP) Hsabx-AflII was integrated into a double digestion product of AflII and XbaI by ligation and transformed into E. coli DH5α to obtain a secretory carrier protein (PDI1 (SP) -Hsabx) and mutant hTF (N413Q / PSL6PDI1 (SP) Hsabx-hTF (N413QN611Q) (8749 bp, FIG. 1), which is a chromosomal single locus integration expression vector of a fusion protein of N611Q) protein, was prepared.

<Production of A8 strain>
S. Leucine uracil auxotrophic S. pombe ^(leu1 - ura4 ^-) strain (genotype: h- leu1-32 ura4-D18) with respect to a is ARC010 strain by Latour method, PSP3 gene, ISP 6 gene, Ppp53 gene, PPP 16 gene , Ppp22 gene, sxa2 gene, ppp80 gene, and ppp20 gene, A8 strain (genotype: h-leu1-32 psp3-D13 isp6-D14 oma1-D10 ppp16-D20 fma2-D13 sxa2-D15 aap1- D17 ppp80-D11) was prepared. The A8 strain was a leucine-requiring strain. More specifically, the Latour method deletes a gene deletion fragment (Latour fragment: a homologous recombination region at both ends, a ura4 ⁺ marker gene sandwiched between it and an OL (overlapping sequence) region) on the fission yeast genome. A latent strain was produced by integrating the gene in the upstream or downstream of the target region by homologous recombination. Subsequently, the latent strain was cultured in a medium containing 5′-FOA, homologous recombination was caused between the incorporated OLs, and a gene-deleted strain in which the deletion target region containing the ura4 ⁺ marker gene was dropped was formed as a colony. . The obtained gene-deleted strain was confirmed by PCR that the target gene was deleted. In this method, since no foreign gene containing the ura4 ⁺ marker gene remains, only the target gene can be deleted without incorporating the foreign gene, and the gene can be deleted repeatedly while recycling the ura4 ⁺ marker gene. Table 1 shows the base sequences of the primers used in preparing a Latour fragment for deleting each gene by PCR.

<Preparation of A8-gas2Δ strain>
The A8-gas2Δ strain (genotype: h-leu1-32 psp3-D13 isp6-D14 oma1-D10 ppp16-D20 fma2-D13 sxa2-D15 aap1-D17 deleted from the Gas2 gene by the Latour method was used for the A8 strain.
ppp80-D11 gas2-D15) was prepared. The obtained gene-deleted strain was confirmed by PCR that the target gene was deleted. The A8-gas2Δ strain was a leucine-requiring strain. Table 2 shows the base sequences of the primers used in preparing a Latour fragment for deleting the Gas2 gene by PCR.

About the produced A8 strain and A8-gas2Δ strain, growth evaluation (measurement of μmax and growth curve) was performed at a test tube level (conditions in which 5 mL of YES was inoculated and cultured at 30 ° C. for 68 hours). As a result, in both strains, the maximum specific growth rate (μmax relative value) indicating the initial rise of the cell growth is slightly decreased (about 8 to 12), compared to the wild strain (ARC001) having the same auxotrophy (leu1 ⁻ ). However, the final cell density (OD ₆₆₀ ) slightly increased (increased by about 9 to 17%), and both strains were confirmed to be able to grow in the same manner as the leucine-requiring wild strain. (Not shown). In particular, no changes in growth and phenotype were observed for the A8 strain and the A8-gas2Δ strain.

<Preparation of 1-copy expression strain of hTF (N413Q / N611Q) gene>
pSL6PDI1 (SP) Hsabx-hTF (N413QN611Q) was cleaved with the restriction enzyme NotI to prepare a vector fragment (6993 bp), and the vector fragment was ligated to the leucine-requiring S. cerevisiae using the lithium acetate method. Transformation was performed using the pombe strain A8 or A8-gas2Δ. The bacterial cells after the transformation treatment were applied to an MMA plate, and colonies formed by complementing leucine requirement were obtained as recombinant clones. Thereafter, a clone confirmed that the target gene was correctly introduced by PCR was selected as a positive clone (hTF (N413Q / N611Q) expression strain). Transformants obtained by introducing pSL6PDI1 (SP) Hsabx-hTF (N413QN611Q) into the A8 strain and the A8-gas2Δ strain were named A8-hTF (1) strain and A8-gas2Δ-hTF (1) strain, respectively.

<Secret expression of 1-copy expression strain of hTF (N413Q / N611Q)>
The A8-hTF (1) strain and the A8-gas2Δ-hTF (1) strain were cultured, and hTF (N413Q / N611Q) was secreted and expressed. Specifically, after culturing for about 24 hours in 5 mL of YES using a glass test tube and performing pre-culture, 5 mL of YPD + MES medium (0.3 M MES buffer was added to YPD) using a glass test tube. The mixture was cultured with shaking in the contained liquid medium (pH 6.0) at 32 ° C. for 3 days or 5 days. The cell density (final reached OD ₆₆₀ ) at the end of the culture was 30 to 36. SDS-PAGE analysis was performed on each of the culture supernatant fraction and the cell pellet collected from the culture to confirm that hTF (N413Q / N611Q) was expressed inside and outside the cell (not shown). . Further, in both the A8-hTF (1) strain and the A8-gas2Δ-hTF (1) strain, the amount of hTF (N413Q / N611Q) contained in the cell pellet was greater than that cultured for 3 days than that cultured for 3 days. The amount of hTF (N413Q / N611Q) in the culture supernatant was high and the amount of hTF (N413Q / N611Q) secreted was increased by extending the culture time. It was found that can be reduced.

<Results of 1-copy expression strain of hTF (N413Q / N611Q)>
The A8-gas2Δ-hTF (1) strain was cultured to secrete and express hTF (N413Q / N611Q). Specifically, after culturing for about 24 hours in 5 mL of YES using a glass test tube and performing pre-culture, in a 5 mL of adenine-containing YPD + MES medium (pH 6.0) using a 24-well plate, Cultured at 32 ° C for 3 days, 4 days, or 5 days with shaking, and subjected to ELISA using a commercially available kit (product name: “Human Transferrin ELISA Quantitation Set”, manufactured by Bethyl Laboratories). Culture supernatant after completion of culture The protein amount of hTF (N413Q / N611Q) secreted into the medium was measured. The longer the culture time, the greater the amount of hTF (N413Q / N611Q) secreted. Moreover, the secretion amount of hTF (N413Q / N611Q) per culture supernatant for 5 days in the culture was 30 to 40 mg / L for the A8-gas2Δ-hTF (1) strain.

<High-density culture of 1-copy expression strain of hTF (N413Q / N611Q)>
The A8-hTF (1) strain and the A8-gas2Δ-hTF (1) strain were cultured, and hTF (N413Q / N611Q) was cultured at high density for secretion expression. Specifically, on the condition of adjusting the pH of the culture solution to 5.5 to 6.0 while controlling the aeration rate of the culture tank, the stirring rate, and the addition rate of the fed-batch medium on a 5 L culture scale. Medium and fed-batch culture were performed. As the liquid medium, an adenine-containing semi-synthetic medium (several percent of Bacto-yeast extract, each synthetic component such as glucose, vitamins, minerals and inorganic salts) was used. While suppressing the glucose concentration to 2%, the medium was continuously added (fed) according to the growth rate and nutrient consumption rate. Eventually, the cell density (OD ₆₆₀ ) was about 200 to 800.

  FIG. 2 shows the result of sampling the culture solution over time and performing SDS-PAGE. 5 μL of the culture solution was applied to each lane. In the figure, “hTF” indicates a band of hTF (N413Q / N611Q). As shown in the CBB-stained image of FIG. 2, the expression of gas2 was suppressed in the A8-gas2Δ-hTF (1) strain at any of the culture times 48, 72 and 96 hours, and the A8-gas was more suppressed than the A8-hTF (1) strain. It was confirmed that the gas2Δ-hTF (1) strain had a higher secretion amount of hTF (N413Q / N611Q).

Further, ELISA was performed in the same manner as described above using the sampled culture solution, and the protein amount of hTF (N413Q / N611Q) secreted into the culture supernatant was measured. As shown in FIG. 3, there is no difference in the amount of secretion between the two strains for about 48 hours from the start of the culture, but after that, the A8-gas2Δ-hTF (1) strain is clearer than the A8-hTF (1) strain. The amount of hTF (N413Q / N611Q) secreted was large. Table 3 shows the final amount of secretion of both strains (A8-hTF (1) strain is 168 hours after the start of culture and A8-gas2Δ-hTF (1) strain is 96 hours after the start of culture). The A8-gas2Δ-hTF (1) strain has a hTF secretion amount of 4.8 times or more per culture medium and a hTF secretion amount per microbial cell (per OD ₆₆₀ ) than the A8-hTF (1) strain. It was more than 1 time.

  Cells were removed from the culture solution after high-density culture by centrifugation, and the collected culture supernatant was subjected to DEAE chromatography to purify hTF. As the DEAE column, a column (product name: “XK26”, manufactured by GE Healthcare Bioscience) packed with DEAE resin (DEAE Sepharose FF (Fast Flow)) was used. Specifically, 900 mL of the culture supernatant was applied to a DEAE column (1 CV = 60.6 mL) equilibrated with an equilibration buffer (20 mM Tris-HCl, pH 8.5), and then eluted buffer B (20 mM Tris- HCl, 1M NaCl, pH 8.5) was applied and eluted with gradient conditions from 0% (15 CV) to 100% (5 CV). The fraction size was 30.3 mL.

FIG. 4 shows the result of SDS-PAGE performed by collecting fractions containing the hTF (N413Q / N611Q) protein (the third to sixth fractions) together. In FIG. 4, “After Gas2 deletion” is a CBB-stained image of the purified fraction of the culture solution of A8-gas2Δ-hTF (1) strain, and “Before Gas2 deletion” is the culture solution of A8-hTF (1) strain. It is a CBB dyeing | staining image of a refinement | purification fraction. As shown in the CBB stained image in FIG. 4, only the band of hTF (N413Q / N611Q) protein was detected in the purified fraction of the culture solution of the A8-gas2Δ-hTF (1) strain, and only one DEAE chromatography was performed. It was confirmed that the hTF (N413Q / N611Q) protein was separated from other proteins and purified with high purity. On the other hand, in the purified fraction of the culture solution of the A8-hTF (1) strain, a plurality of bands were detected on the polymer side in addition to the hTF (N413Q / N611Q) protein. Of these, the band near 115 kDa was cut out and subjected to N-terminal sequence analysis, and was confirmed to be Gas2. Since the secretion of Gas2 was not clearly confirmed in the culture at a normal cell density, it was considered to be a phenomenon peculiar to high-density culture. In the A8-hTF (1) strain, in addition to hTF (N413Q / N611Q), the amount of secreted production of Gas2 also increases, so that the amount of secreted production of hTF (N413Q / N611Q) is higher than that of the A8-gas2Δ-hTF (1) strain. It was suggested that was kept low.
It should be noted that the entire content of the specification, claims, abstract and drawings of Japanese Patent Application No. 2006-003606 filed on Jan. 12, 2016 is cited herein as the disclosure of the specification of the present invention. Incorporated.

Claims (15)

  Schizo Saccharomyces pombe as a host, comprising a transferrin gene, and a secretory signal peptide gene that expresses transferrin bound to a secretory signal peptide that exists in the upstream region of the transferrin gene and functions in the host, A transformant in which the Gas2 gene originally possessed by is deleted or inactivated.   The transformant according to claim 1, wherein the transferrin gene is a gene encoding a natural TF protein derived from a mammal or a modified protein thereof.   The transformant according to claim 1 or 2, wherein the transferrin gene is a gene encoding human transferrin or a modified protein thereof.   The transferrin gene is a mutant transferrin gene in which a mutation is introduced into a gene encoding a natural transferrin possessed by a mammal, and the mutant transferrin gene is at least one N-linked sugar chain modification site of the natural transferrin The transformant according to claim 1 or 2, which is a gene encoding a mutant transferrin in which the asparagine residue is deleted or substituted with another amino acid residue.   The transformant according to claim 4, wherein the transferrin gene is a mutant transferrin gene in which a mutation is introduced into a gene encoding a natural transferrin possessed by a human.   The transformant according to any one of claims 1 to 5, wherein at least one protease gene originally possessed by the host is deleted or inactivated.   The transformant according to claim 6, wherein the protease gene is a gene selected from the group consisting of a metalloprotease gene group, a serine protease gene group, a cysteine protease gene group, and an aspartic protease gene group.   The transformant according to claim 6 or 7, wherein the protease gene is a gene selected from the group consisting of psp3 gene, isp6 gene, ppp53 gene, ppp16 gene, ppp22 gene, sxa2 gene, ppp80 gene and ppp20 gene.   The transformant according to any one of claims 1 to 8, wherein the transferrin gene is directly or indirectly linked downstream of a gene encoding a secretory carrier protein containing a secretory signal peptide.   A gene encoding the secretory signal peptide part of the host PDI1, a gene encoding the ab domain part of human PDI1, and a gene encoding the x domain part of the host PDI1 The transformant according to claim 9, which is a fusion gene.   A method for producing transferrin, comprising culturing the transformant according to any one of claims 1 to 10 in a liquid medium and obtaining transferrin from the liquid medium.   The method for producing transferrin according to claim 11, wherein the transformant is cultured in a liquid medium having a pH of 5.5 to 6.5.   The method for producing transferrin according to claim 11 or 12, wherein the transformant is cultured in a liquid medium containing adenine. The method for producing transferrin according to any one of claims 11 to 13, wherein the transformant is cultured until the bacterial cell density (OD ₆₆₀ ) reaches 100 or more, and then transferrin is obtained from the liquid medium.   Schizo Saccharomyces pombe as a host, incorporating a secretory signal peptide gene functioning in the host and a transferrin gene located downstream of the secretory signal peptide gene, and deleting the Gas2 gene originally possessed by the host or A method for producing a transformant, wherein the transformant is inactivated.

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